US8097254B2 - Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis - Google Patents

Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis Download PDF

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US8097254B2
US8097254B2 US10/821,930 US82193004A US8097254B2 US 8097254 B2 US8097254 B2 US 8097254B2 US 82193004 A US82193004 A US 82193004A US 8097254 B2 US8097254 B2 US 8097254B2
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antibody
angiogenesis
antibodies
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US20060133994A1 (en
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Dario Neri
Lorenzo Tarli
Francesca Viti
Manfred Birchler
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Eidgenoessische Technische Hochschule Zurich ETHZ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C63/00—Compounds having carboxyl groups bound to a carbon atoms of six-membered aromatic rings
    • C07C63/04—Monocyclic monocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to antibodies with sub-nanomolar affinity specific for a characteristic epitope of the ED-B domain of fibronectin, a marker of angiogenesis. It also relates to the use of radiolabelled high-affinity anti-ED-B antibodies for detecting new-forming blood vessels in vivo and a diagnostic kit comprising said antibody.
  • the invention refers to conjugates comprising the above said antibodies and a suitable photoactive molecule (e.g., a photosensitizer) and to their use in the detection and/or coagulation of new blood vessels.
  • a suitable photoactive molecule e.g., a photosensitizer
  • angiogenesis a correlation between microvessel density and tumour invasiveness has been reported for a number of tumours (Folkman (1995). Nature Med., 1, 27-31). Moreover, angiogenesis underlies the majority of ocular disorders which result in loss of vision [Lee et al., Surv. Ophthalmol. 43, 245-269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9764-9769 (1996).].
  • Molecules capable of selectively targeting markers of angiogenesis would create clinical opportunities for the diagnosis and therapy of tumours and other diseases characterised by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration.
  • Markers of angiogenesis are expressed in the majority of aggressive solid tumours and should be readily accessible to specific binders injected intravenously (Pasqualini et al. (1997). Nature Biotechnol., 15, 542-546; Neri et al. (1997), Nature Biotechnol., 15 1271-1275).
  • Targeted occlusion of the neovasculature may result in tumour infarction and collapse (O'Reilly et al. (1996). Nature Med., 2, 689-692; Huang et al. (1997). Science, 275, 547-550).
  • fibronectin a sequence of 91 aminoacids identical in mouse, rat and human, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neo-vascular structures (Castellani et al. (1994). Int. J. Cancer 59, 612-618) and could represent a target for molecular intervention. Indeed, we have recently shown with fluorescent techniques that anti-ED-B single-chain Fv antibody fragments (scFv) accumulate selectively in tumoural blood vessels of tumour-bearing mice, and that antibody affinity appears to dictate targeting performance (Neri et al. (1997). Nature Biotechnol., 15 1271-1275; International Patent Application No. PCT/GB97/01412, based on GB96/10967.3). Tumour targeting was evaluated 24 hours after injection, or at later time points.
  • Peters et al. disclose polyclonal antibodies raised to antigens containing no FN sequence other than the intact ED-B domain and show that they bind specifically and directly to this domain.
  • JP02076598 and JP04169195 refer to anti-ED-B antibodies. It is not clear from these documents if monoclonal anti ED-B antibodies are described. Moreover, it seems impossible that a single antibody (such as the antibody described in JP02076598) has “an antigen determinant in amino acid sequence of formulae (1), (2) or (3):
  • a monoclonal antibody should recognize a well-defined epitope.
  • the BC1 antibody described by Carnemolla et al. 1992, J. Biol. Chem. 267, 24689-24692 recognises an epitope on domain 7 of FN, but not on the ED-B domain, which is cryptic in the presence of the ED-B domain of fibronectin. It is strictly human-specific. Therefore, the BC1 antibody and the antibodies of the present invention show different reactivity. Furthermore, the BC1 antibody recognises domain 7 alone, and domain 7-8 of fibronectin in the absence of the ED-B domain (Carnemolla et al. 1992, J. Biol. Chem. 267, 24689-24692). Such epitopes could be produced in vivo by proteolytic degradation of FN molecules.
  • the advantage of the reagents according to the present invention is that they can localise on FN molecules or fragments only if they contain the ED-B domain.
  • immunoscintigraphy is one of the techniques of choice.
  • patients are imaged with a suitable device (e.g., a gamma camera), after having been injected with radiolabelled compound (e.g., a radionuclide linked to a suitable vehicle).
  • radiolabelled compound e.g., a radionuclide linked to a suitable vehicle.
  • radiolabelled compound e.g., a radionuclide linked to a suitable vehicle.
  • short-lived gamma emitters such as technetium-99m, iodine-123 or indium-111 are typically used, in order to minimise exposure of the patient to ionising radiations.
  • the most frequently used radionuclide in Nuclear Medicine Departments is technetium-99m (99 mTc), a gamma emitter with half-life of six hours.
  • 99 mTc technetium-99m
  • Patients injected with 99 mTc-based radiopharmaceuticals can typically be imaged up to 12-24 hours after injections; however, accumulation of the nuclide on the lesion of interest at earlier time points is desirable.
  • a further object of the present invention is to provide radiolabelled antibodies in suitable format, directed against the ED-B domain of fibronectin, that detect tumour lesions already few hours after injection.
  • these objects are achieved by an antibody with specific affinity for a characteristic epitope of the ED-B domain of fibronectin and with improved affinity to said ED-B epitope.
  • the above described antibody is used for rapid targeting markers of angiogenesis.
  • Another aspect of the present invention is a diagnostic kit comprising said antibody and one or more reagents for detecting angiogenesis.
  • Still a further aspect of the present invention is the use of said antibody for diagnosis and therapy of tumours and diseases which are characterized by vascular proliferation.
  • conjugates comprising said antibodies and a suitable photoactive molecules (e.g. a judiciously chosen photosensitizer), and their use for the selective light-mediated occlusion of new blood vessels.
  • a suitable photoactive molecules e.g. a judiciously chosen photosensitizer
  • immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.
  • antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv; dAb, Fd; and diabodies. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody.
  • Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400.
  • a hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of a whole antibody can perform the function of binding antigens.
  • binding fragments are (I) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g.
  • antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies these may be conventional bispecific antibodies, which can be manufactured in a variety of ways Holliger and Winter (1993), Curr. Opin. Biotech., 4, 446-449), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.
  • Other forms of bispecific antibodies include the single-chain CRAbs described by Neri et al. ((1995) J. Mol. Biol., 246, 367-373).
  • CDRs complementarity-determining regions
  • variant refers to a molecule (the variant) which although having structural differences to another molecule (the parent) retains some significant homology and also at least some of the biological function of the parent molecule, e.g. the ability to bind a particular antigen or epitope.
  • Variants may be in the form of fragments, derivatives or mutants.
  • a variant, derivative or mutant may be obtained by modification of the parent molecule by the addition, deletion, substitution or insertion of one or more aminoacids, or by the linkage of another molecule. These changes may be made at the nucleotide or protein level.
  • the encoded polypeptide may be a Fab fragment which is then linked to an Fc tail from another source.
  • a marker such as an enzyme, fluorescein, etc, may be linked.
  • a functionally equivalent variant form of an antibody “A” against a characteristic epitope of the ED-B domain of fibronectin could be an antibody “B” with different sequence of the complementarity determining regions, but recognising the same epitope of antibody “A”.
  • the high-affinity antibody L19 and D1.3 (an antibody specific for an irrelevant antigen, hen egg lysozyme) were radiolabelled and injected in tumour-bearing mice. Tumour, blood and organ biodistributions were obtained at different time points, and expressed as percent of the injected dose per gram of tissue (% ID/g). Already 3 hours after injection, the % ID/g (tumour) was better than the % ID/g (blood) for L19, but not for the negative control D1.3. The tumour:blood ratios increased at longer time points. This suggests that the high-affinity antibody L19 may be a useful tumour targeting agent, for example for immunoscintigraphic detection of angiogenesis.
  • a photosensitiser could be defined as a molecule which, upon irradiation and in the presence of water and/or oxygen, will generate toxic molecular species (e.g., singlet oxygen) capable of reacting with biomolecules, therefore potentially causing damage to biological targets such as cells, tissues and body fluids.
  • toxic molecular species e.g., singlet oxygen
  • Photosensitisers are particularly useful when they absorb at wavelengths above 600 nm. In fact, light penetration in tissues and body fluids is maximal in the 600-900 nm range [Wan et al. (1981) Photochem. Photobiol. 34, 679-681).
  • the high-affinity L19 antibody specific for the ED-B domain of fibronectin selectively localises to newly formed blood vessels in a rabbit model of ocular angiogenesis upon systemic administration.
  • the L19 antibody chemically coupled to the photosensitising agent tin (IV) chlorin e 6 and irradiated with red light, mediated the selective occlusion of ocular neovasculature and promoted apoptosis of the corresponding endothelial cells.
  • photosensitisers are among the few species capable of mediating their toxic activity in the immediate proximity of the antibody to which they have been conjugated.
  • alpha-emitting radionuclides such as astatine-211, bismuth-212 and bismuth-213, which produce energetic alpha particles having a range in tissue penetration of 50-100 ⁇ M (Hauck et al. 1998, Brit. J. Cancer 77, 753-759), depositing energy that is about 500 times greater than that of beta particles, e.g. yttrium-90 (100 KeV/ ⁇ M vs. 0.2 KeV/ ⁇ M, respectively) (McDevitt et al. 1998, Eur. J. Nucl. Med. 25, 1341-135).
  • Photosensitisers or alpha-emitting radionuclides conjugated to the antibodies specific for the ED-B domain of fibronectin described in the present application, as for example L 19, can be targeted to new blood vessels in vivo, as illustrated in the examples hereinafter reported. Thanks to this extraordinar specificity of localisation and to the short range of action of the toxic species released by photosensitiser and alpha-emitting radionuclides, specific damage can be inferred in the immediate proximity of the labelled antibody, while sparing adjacent tissues/cells.
  • Toxic species with a short path of action (such as singlet oxygen and alpha particles), when delivered by the L19 antibody, offer the possibility to selectively damage the endothelial cells of new blood vessels, while sparing normal tissues and blood cells.
  • This level of selectivity should offer tremendous advantages for the control of angiogenesis-related disorders, such as cancer, rheumatoid arthritis, neo-vasculature associated ocular disorders and psoriasis.
  • the therapeutic advantage of the radiolabelled antibody will be related to the ratio between the total dose of radioactivity delivered by the antibody to the tumour and the total dose of radioactivity delivered by the antibody to blood or organs, normalised per gram of fluid or tissue (Behr et al. 1998, Int. J. Cancer 77, 787-795).
  • the dose of radioactivity delivered per gram of tumour is proportional to the area under the curve (AUC) of the % ID/g (tumour), plotted versus time.
  • the dose of radioactivity delivered to blood is proportional to the area under the curve (AUC) of the % ID/g (blood), plotted versus time.
  • AUC area under the curve
  • the ratio of AUC (tumour)/AUC (blood) is equal to 3.6 ( FIG. 12 ). This ratio may somewhat increase when the AUCs are measured for longer time periods, but therapeutic ratios greater than 6-7 are rarely achieved.
  • a short-range-acting toxic species e.g., a photosensitiser or an alpha-particle emitting radionuclide
  • the diffusive toxic species e.g., singlet oxygen or an alpha particle
  • new-forming blood vessels in most tumours constitute less than 2% of the total tumour mass
  • the relevant parameter for predicting therapeutic benefit will be the ratio AUC(vessel)/AUC(blood).
  • astatine-211 as a particularly interesting alpha-particle emitting radionuclide for biomedical application.
  • the half-life of its radioactive decay is considerably longer than the ones of bismuth-212 and bismuth-213 (7.2 hrs, compared to 1.0 hr and 45 min., respectively) (Larsen and Bruland 1998, Brit. J. Cancer 77, 1115-1122).
  • Astatine is the heaviest halogen and no stable isotopes of this element exist. Since it is directly below iodine in the periodic table, it might be expected that the two halogens would possess similar chemical properties.
  • the bifunctional m-MeATE agent can be used both to label L19 with the gamma-emitting iodine-125 and the alpha-emitting astatine-211 in a two-step methodology.
  • Protein labeling with iodine-125 through m-MeATE rather than conventional electrophilic methods is reported to provide protein iodination sites which are more inert towards dehalogenation in vivo (Garg et al. 1989, Appl. Radiat. Isot. 40, 485-490).
  • the labeling protein coupling efficiency together with the conjugate immunoreactivity arising from this two-step radiolabeling methodology were studied through the reaction with iodine-125.
  • m-MeATE and t-buthylhydroperoxide were added to Na 125 I.
  • the radioiodinated product N-succinimidyl-3-( 125 I)-benzoate was isolated and separated from unconjugated m-MeATE by chromatography using a disposable silica gel Sep-Pak column and a gradient of ethyl acetate in hexane as eluent phase. Starting from 100 ⁇ Ci of Na 125 I, we coupled 90 ⁇ Ci of 125 I to the N-succinimidyl benzoate.
  • Astatine-211 may offer the additional advantage that it is likely to be detached from L19 by dehalogenases in the kidney glomeruli (T. M. Behr et al. 1999, Cancer Res. 59, 2635-43), but not in tumoural vessels, therefore reducing kidney toxicity of the radiolabelled compound in spite of the clearance of the immunoconjugate via the renal route.
  • glomeruli in the kidneys will not be damaged, since for most applications irradiation of the kidneys is not foreseen.
  • conjugates consisting of the antibody linked to a photosensitiser and a radionucleotide in order to take advantages of the activity of both.
  • DNA encoding antibody scFv L-19 has been deposited on Sep. 25, 2008, in ATCC (Manassas, Va.), and has accession no. PTA-9529.
  • FIG. 1 shows a designed antibody phage library.
  • VH primers are disclosed as SEQ ID NOS 11-14, respectively, in order of appearance.
  • VL primers are disclosed as SEQ ID NOS 15-18, respectively, in order of appearance;
  • FIG. 2 shows 2D gels and Western blotting of a lysate of human melanoma COLO-38 cells
  • FIG. 3 shows immunohistochemical experiments of glioblastoma multiforme
  • FIG. 4 shows an analysis of the stability of antibody-(ED-B) complexes.
  • FIG. 5 shows biodistribution of tumour bearing mice injected with radiolabelled antibody fragments.
  • FIG. 6 shows rabbit eyes with implanted pellet
  • FIG. 7 shows immunohistochemistry of rabbit cornea sections.
  • FIG. 8 shows the immunohistochemistry of sections of ocular structures of rabbits (cornea, iris and conjunctiva) using a red alkaline phosphatase substrate and hematoxylin.
  • FIG. 9 shows the localisation of fluorescently-labelled antibodies in ocular neovasculature.
  • FIG. 10 shows the macroscopic appearance of the eyes of rabbits injected with proteins coupled to photosensitizers, before and after irradiation.
  • FIG. 11 shows the microscopic analysis of sections of ocular structures of rabbits injected with proteins coupled to photosensitizers and irradiated with red light.
  • FIG. 12 shows schematic diagrams in which the % of injected dose delivered per gram, respectively of blood and tumour, are plotted versus time.
  • FIG. 13 shows the accumulation of L19 around new vessels.
  • FIG. 14 shows schematic diagrams reporting the relevant % of injected doses per gram, in the case of the L19 antibody coupled to an .alpha.- and .beta.-emitter respectively, plotted versus time.
  • FIG. 15 shows the labelling method according to the Garg protocol modified as described hereinafter.
  • FIGS. 16 a and 16 b report the 1H-NMR spectrum of 3-(trimethylstannyl)-benzoic acid.
  • FIGS. 17 a and 17 b report the 1H-NMR spectrum of N-succinimidyl-3-(trimethylstannyl)-benzoate
  • FIG. 18 shows the EI-MS of N-succinimidyl-3-(trimethylstannyl)-benzoate.
  • FIG. 1 shows:
  • FIG. 1A Antibody fragments are displayed on phage as p111 fusion, as schematically depicted.
  • the Vk CDRs backbone and the VH CDR backbone are shown.
  • Residues subject to random mutation are Vk CDR3 positions 91, 93, 94 and 96, and VH CDR3 positions 95, 96, 97, and 98.
  • the Cb atoms of these side chains are shown in darker shades.
  • Also shown (in grey) are the residues of CDR1 and CDR2, which can be mutated to improve antibody affinity.
  • FIG. 1B PCR amplification and library cloning strategy.
  • the DP47 and DPK22 germline templates were modified (see text) to generate mutations in the CDR3 regions. Genes are indicated as rectangles, and CDRs as numbered boxes within the rectangle.
  • the VH and the VL segments were then assembled and cloned in pDN332 phagemid vector. Primers used in the amplification and assembly are listed at the bottom.
  • FIG. 2 shows
  • FIG. 3 shows:
  • FIG. 4 shows:
  • FIG. 4A BIAcore sensograms, showing the improved dissociation profiles obtained upon antibody affinity-maturation.
  • FIG. 4B Native gel electrophoretic analysis of scFv-(ED-B) complexes. Only the high-affinity antibody L19 can form a stable complex with the fluorescently labeled antigen. Fluorescence detection was performed as described (Neri et al. (1996) BioTechniques, 20, 708-712).
  • FIG. 4C Competition of the scFv-(ED-B-biotin) complex with a 100-fold molar excess of unbiotinylated ED-B, monitored by electrochemiluminescence using an Origen apparatus. A long half-life for the L19-(ED-B) complex can be observed. Black squares: L19; Open triangles: H10.
  • FIG. 5 shows:
  • Tumour and blood biodistributions are plotted versus time. Relevant organ biodistributions is also reported.
  • FIG. 6 shows rabbit eyes with implanted polymer pellets soaked with angiogenic substances.
  • FIG. 7 shows immunohistochemistry of sections of rabbit cornea with new-forming blood vessels, stained with the L19 antibody.
  • FIG. 8 shows immunohistochemical studies of ocular structures using the L19 antibody. A specific red staining is observed around neovascular structures in the cornea ( FIG. 8 a ), but not around blood vessels in the iris ( FIG. 8 b ) and in the conjunctiva ( FIG. 8 c ). Small arrows: corneal epithelium. Relevant blood vessels are indicated with large arrows. Scale bars: 50 ⁇ m
  • FIG. 9 shows immunophotodetection of fluorescently labelled antibodies targeting ocular angiogenesis.
  • a strongly fluorescent corneal neovascularisation (indicated by an arrow) is observed in rabbits injected with the antibody conjugate L19-Cy5 ( FIG. 9 a ), specific for the ED-B domain of FN, but not with the antibody HyHEL-10-Cy5 ( FIG. 9 b ).
  • Immunofluorescence microscopy on cornea sections confirmed that L19-Cy5 ( FIG. 9 c ), but not HyHEL-10-Cy5 ( FIG. 9 d ) localises around neovascular structures in the cornea.
  • Images ( FIG. 9 a,b ) were acquired 8 h after antibody injection; ( FIG. 9 c,d ) were obtained using cornea sections isolated from rabbits 24 h after antibody injection. P, pellet.
  • FIG. 10 shows macroscopic images of eyes of rabbits treated with photosensitiser conjugates. Eye of rabbit injected with L19-PS before ( FIG. 10 a ) and 16 h after irradiation with red light ( FIG. 10 b ). The arrow indicates coagulated neovasculature, which is confirmed as a hypofluorescent area in the Cy5 fluoroangiogram of panel ( FIG. 10 c ) 16 h after irradiation. Note that no coagulation is observed in other vascular structures, for example in the dilated conjuctival vessels. For comparison, a Cy5 fluoroangiogram with hyperfluorescence of leaky vessels, and the corresponding colour photograph of untreated rabbit eye are shown in ( FIG.
  • FIGS. 10 e , 10 f , 10 g are analogous to ( FIGS. 10 a , 10 b , 10 c ), but correspond to a rabbit injected with ovalbumin-PS and irradiated with red light. No coagulation can be observed, and the angiogram reveals hyperfluorescence of leaky vessels. The eyes of rabbits with early-stage angiogenesis and injected with L19-PS are shown in ( FIGS. 10 i , 10 l ). Images before ( FIG. 10 i ) and 16 h after irradiation with red light ( FIG. 10 j ) reveal extensive and selective light-induced intravascular coagulation (arrow).
  • Vessel occlusion is particularly evident in the irradiated eye ( FIG. 10 l ) of a rabbit immediately after euthanasia, but cannot be detected in the non-irradiated eye ( FIG. 10 k ) of the same rabbit.
  • P pellet.
  • Arrowheads indicate the corneo-scleral junction (limbus). In all figures, dilated pre-existing conjunctival vessels are visible above the limbus, whereas growth of corneal neovascularisation can be observed from the limbus towards the pellet (P).
  • FIG. 11 shows microscopic analysis of selective blood vessel occlusion.
  • H/E sections of corneas (a,e,b,f: non-fixed; i,j: paraformaldehyde fixed) of rabbits injected with ovalbumin-PS ( FIGS. 11 a , 11 e , 11 i ) or L19-PS ( FIGS. 11 b , 11 f , 11 j ) and irradiated.
  • Large arrows indicate representative non damaged ( FIGS. 11 e , 11 i ) or completely occluded ( FIGS. 11 f , 11 j ) blood vessels.
  • FIGS. 11 b , 11 f , 11 j In contrast to the selective occlusion of corneal neovasculature and restricted perivascular damage (eosinophilia) mediated by L19-PS after irradiation ( FIGS. 11 b , 11 f , 11 j ), vessels in the conjunctiva ( FIG. 11 k ) and iris ( FIG. 11 l ) do not show sign of damage in the same rabbit. Fluorescent TUNEL assay indicates the different number of apoptotic cells in sections of irradiated rabbits injected with L19-PS ( 11 c , 11 g ) or with ovalbumin-PS ( 11 d , 11 h ). Large arrows indicate some relevant vascular structures. Small arrows indicate corneal epithelium. Scale bars: 100 ⁇ m ( 11 a - 11 d ) and 25 ⁇ m ( 11 e - 11 l )
  • FIG. 12 shows the area under the curve (AUC) of the radioactivity delivered by scFv(L19) to the blood and to the tumour during the first 24 hours after intravenous injection.
  • AUC area under the curve
  • FIG. 13 shows microautoradiographic analysis of an F9 teratocarcinoma dissected from a nude mouse, after injection of radiolabelled scFv(L19). The pictures show that scFv(L19) accumulates around vascular structure but not in the surrounding normal mouse tissue.
  • FIG. 14 illustrates a schematic diagram of the radiommunotherapy performed with the anti-angiogenesis scFv(L19) coupled with .beta.- or .alpha.-emitting radionuclide.
  • a beta emitter e.g., Yttrium-90
  • the majority of the targeted tumoural area is irradiated, since these .beta.-particles have a range in tissue of several millimeters.
  • scFv(L19) to an alpha emitter (e.g. Astatine-211 or Bismuth-212 or Bismuth-213), the radiation is deposited only around the targeted tumoural blood vessels (penetration: few dozens of micrometers).
  • the relevant parameters for therapeutic efficacy is the vessel:blood ratio of the percent injected dose of radioactivity per gram of tissue, rather than the tumour:blood ratio (which is the relevant parameter for beta emitting nuclides).
  • FIG. 15 shows the procedure for labelling scFv(L19) with Iodine-125 and Astatine-211 using the N-succinimidyl 3-(trimethylstannyl)benzoate (m-MeATE) synthesized from m-bromobenzoic acid.
  • FIG. 16 a shows the 1 H-NMR spectrum of 3-(trimethylstannyl)benzoic acid in CDCl 3 .
  • the chemical shifts (ppm) are indicated in the X-axis.
  • FIG. 16 b shows the chemical shift in ppm of the protons in the aromatic moiety located at low magnetic field of the NMR spectrum.
  • FIG. 17 a shows the 1 H-NMR spectrum of m-MeATE in CDCl 3 .
  • the relative values of peak intensity are reported between the X-axis reporting the chemical shift (ppm) and the spectrum baseline.
  • FIG. 17 b shows an enlarged portion of the 1 H-NMR spectrum of m-MeATE.
  • FIG. 18 shows the electron ionization mass spectrum (EI-MS) of m-MeATE (molecular weight 383).
  • the mass to charge value (m/e) of 368 represents the mass of the molecular ion (M + )-15 (CH 3 ).
  • VH human antibody library
  • Vk Vk22; Cox et al. (1994). Eur. J. Immunol., 24, 827-836 germline genes (see FIG. 1 for the cloning and amplification strategy).
  • the VH component of the library was created using partially degenerated primers ( FIG. 1 ) in a PCR-based method to introduce random mutations at positions 95-98 in CDR3.
  • the VL component of the library was generated in the same manner, by the introduction of random mutations at positions 91, 93, 94 and 96 of CDR3.
  • VH-VL scFv fragments were constructed by PCR assembly ( FIG. 1 ; Clackson et al. (1991). Nature, 352, 624-628), from gel-purified VH and VL segments. 30 ⁇ g of purified VH-VL scFv fragments were double digested with 300 units each of NcoI and NotI, then ligated into 15 ⁇ g of Not1/Nco1 digested pDN332 phagemid vector.
  • pDN332 is a derivative of phagemid pHEN1 (Hoogenboom et al. (1991). Nucl.
  • Transformations into TG1 E. coli strain were performed according to Marks et al. (1991. J. Mol. Biol., 222, 581-597) and phages were prepared according to standard protocols (Nissim et al. (1991). J. Mol. Biol., 222, 581-597). Five clones were selected at random and sequenced to check for the absence of pervasive contamination.
  • Recombinant fibronectin fragments ED-B and 7B89, containing one and four type III homology repeats respectively, were expressed from pQE12-based expression vectors (Qiagen, Chatsworth, Calif., USA) as described (Carnemolla et al. (1996). Int. J. Cancer, 68, 397-405).
  • Phages were incubated with antigen in 2% milk/PBS (MPBS) for 10 minutes.
  • MPBS milk/PBS
  • DTT dithiothreitol
  • the eluted phage was used to infect exponentially-growing HB2151 E. coli cells and plated on (2 ⁇ TY+1% glucose+100 ⁇ g/ml ampicillin) ⁇ 1.5% agar plates. Single colonies were grown in 2 ⁇ TY+0.1% glucose+100 ⁇ g/ml ampicillin, and induced overnight at 30 degrees with 1 mM IPTG to achieve antibody expression.
  • the resulting supernatants were screened by ELISA using streptavidin-coated microtitre plates treated with 10 nM biotinylated-ED-B, and anti-FLAG M2 antibody (IBI Kodak, New Haven, Conn.) as detecting reagent. 32% of screened clones were positive in this assay and the three of them which gave the strongest ELISA signal (E1, A2 and G4) were sequenced and further characterised.
  • ELISA assays were performed using biotinylated ED-B recovered from a gel spot, biotinylated ED-B that had not been denatured, ED-B linked to adjacent fibronectin domains (recombinant protein containing the 7B89 domains), and a number of irrelevant antigens.
  • Antibodies E1, A2 and G4 reacted strongly and specifically with all three ED-B containing proteins. This, together with the fact that the three recombinant antibodies could be purified from bacterial supernatants using an ED-B affinity column, strongly suggests that they recognise an epitope present in the native conformation of ED-B. No reaction was detected with fibronectin fragments which did not contain the ED-B domain (data not shown).
  • Antibodies E1, A2 and G4 were used to immunolocalise ED-B containing fibronectin (B-FN) in cryostat sections of glioblastoma multiforme, an aggressive human brain tumour with prominent angiogenetic processes.
  • FIG. 3 shows serial sections of glioblastoma multiforme, with the typical glomerulus-like vascular structures stained in red by the three antibodies. Immunostaining of sections of glioblastoma multiforme samples frozen in liquid nitrogen immediately after removal by surgical procedures, was performed as described (Carnemolla et al. (1996). Int. J. Cancer, 68, 397-405, Castellani et al. (1994). Int. J. Cancer, 59, 612-618).
  • ScFv(E1) was selected to test the possibility of improving its affinity with a limited number of mutations of CDR residues located at the periphery of the antigen binding site ( FIG. 1A ).
  • the resulting repertoire of 4 ⁇ 10 8 clones was selected for binding to the ED-B domain of fibronectin. After two rounds of panning, and screening of 96 individual clones, an antibody with 27-fold improved affinity was isolated (H10; Table 1).
  • scFv(E1) was PCR amplified with primers LMB1bis (5′-GCG GCC CAG CCG GCC ATG GCC GAG-3′ (SEQ ID NO: 1)) and DP47CDR1for (5′-GA GCC TGG CGG ACC CAG CTC ATM NNM NNM NNGCTA AAG GTG AAT CCA GAG GCT G-3′ (SEQ ID NO: 2)) to introduce random mutations at positions 31-33 in the CDR1 of the VH (for numbering: 28), and with primers DP47CDR1back (5′-ATG AGC TGG GTC CGC CAG GCT CC-3′ (SEQ ID NO: 3)) and DP47CDR2for (5′-GTC TGC GTA GTA TGT GGT ACC MNN ACT ACC MNN AAT MNN TGA GAC CCA CTC CAG CCC CTT-3′ (SEQ ID NO: 4)) to randomly mutate positions
  • the three resulting PCR products were gel purified and assembled by PCR (21) with primers LMB1bis and JforNot (94° C.
  • the resulting single PCR product was purified from the PCR mix, double digested with NotI/NcoI and ligated into NotI/NcoI digested pDN332 vector. Approximately 9 ⁇ g of vector and 3 ⁇ g of insert were used in the ligation mix, which was purified by phenolisation and ethanol precipitation, resuspended in 50 ⁇ l of sterile water and electroporated in electrocompetent TGI E. coli cells. The resulting affinity maturation library contained 4 ⁇ 10 8 clones. Antibody-phage particles, produced as described (Nissim et al. (1994).
  • the gene of scFv(H10) was PCR amplified with primers LMB1bis and DPKCDR1for (5′-G TTT CTG CTG GTA CCA GGC TAA MNN GCT GCT GCT AAC ACT CTG ACT G (SEQ ID NO: 7)) to introduce a random mutation at position 32 in CDR1 of the VL (for numbering: Chothia and Lesk (1987) J. Mol.
  • the remaining portion of the scFv gene was amplified with oligos DPKCDR2back (5′-GCA TCC AGC AGG GCC ACT GGC-3′ (SEQ ID NO: 10)) and JforNot (94 C 1 min, 60 C 1 min, 72 C 1 min)
  • the three resulting products were assembled, digested and cloned into pDN332 as described above for the mutagenesis of the heavy chain.
  • the resulting library was incubated with biotinylated ED-B in 3% BSA for 30 min., followed by capture on a streptavidin-coated microtitre plate (Boehringer Mannheim GmbH, Germany) for 10 minutes.
  • the phages were eluted with a 20 mM DTT solution (1,4-Dithio-DL-threitol, Fluka) and used to infect exponentially growing TG1 cells.
  • Antibody fragments were then eluted with triethylamine 100 mM, immediately neutralised with 1 M Hepes, pH 7, and dialysed against PBS. Affinity measurements by BIAcore were performed with purified antibodies as described (Neri et al. (1997). Nature Biotechnol., 15 1271-1275) [ FIG. 4 ]. Band-shift analysis was performed as described (Neri et al. (1996). Nature Biotechnology, 14, 385-390), using recombinant ED-B fluorescently labelled at the N-terminal extremity (Carnemolla et al. (1996). Int. J. Cancer, 68, 397-405, Neri et al. (1997).
  • anti-ED-B antibodies (30 nM) were incubated with biotinylated ED-B (10 nM) for 10 minutes, in the presence of M2 anti-FLAG antibody (0.5 ⁇ g/ml) and polyclonal anti-mouse IgG (Sigma) which had previously been labelled with a rutenium complex as described (Deaver, D. R. (1995). Nature, 377, 758-760).
  • M2 anti-FLAG antibody 0.5 ⁇ g/ml
  • polyclonal anti-mouse IgG Sigma
  • Radioiodinated scFv(L19) or scFv(D1.3) (an irrelevant antibody specific for hen egg lysozyme) were injected intravenously in mice with subcutaneously implanted murine F9′ teratocarcinoma, a rapidly growing aggressive tumour. Antibody biodistributions were obtained at different time points ( FIG. 4 ). ScFv(L19) and scFv(D1.3) were affinity purified on an antigen column (Neri et al. (1997, Nature Biotechnol. 15, 1271-1273) and radiolabelled with iodine-125 using the Iodogen method (Pierce, Rockford, Ill., USA).
  • Radiolabelled antibody fragments retained >80% immunoreactivity, as evaluated by loading the radiolabelled antibody onto an antigen column, followed by radioactive counting of the flow-through and eluate fractions.
  • Nude mice (12 weeks old Swiss nudes, males) with subcutaneously-implanted F9 murine teratocarcinoma (Neri et al. (1997) Nature Biotechnol. 15, 1271-1273) were injected with 3 ⁇ g (3-4 ⁇ Ci) of scFv in 100 ⁇ l saline solution. Tumour size was 50-250 mg, since larger tumours tend to have a necrotic centre. However, targeting experiments performed with larger tumours (300-600 mg) gave essentially the same results. Three animals were used for each time point.
  • mice were killed with humane methods, and organs weighed and radioactively counted. Targeting results of representative organs are expressed as percent of the injected dose of antibody per gram of tissue (% ID/g). ScFv(L19) is rapidly eliminated from blood through the kidneys; unlike conventional antibodies, it does not accumulate in the liver or other organs. Eight percent of the injected dose per gram of tissue localises on the tumour already three hours after injection; the subsequent decrease of this value is due to the fact that the tumour doubles in size in 24-48 hours. Tumour:blood ratios at 3, 5 and 24 hours after injection were 1.9, 3.9 and 11.8 respectively for L19, but always below 1.0 for the negative control antibody.
  • Anti-ED-B Antibodies Selectively Stain Newly-Formed Ocular Blood Vessels
  • Angiogenesis the formation of new blood vessels from pre-existing ones, is a characteristic process which underlies many diseases, including cancer and the majority of ocular disorders which result in loss of vision.
  • the ability to selectively target and occlude neovasculature will open diagnostic and therapeutic opportunities.
  • B-FN is a specific marker of ocular angiogenesis and whether antibodies recognising B-FN could selectively target ocular neovascular structures in vivo upon systemic administration.
  • angiogenesis in the rabbit cornea, which allows the direct observation of new-blood vessels, by surgically implanting pellets containing vascular endothelial growth factor or a phorbol ester ( FIG. 6 ).
  • the eye was illuminated with a tungsten halogen lamp (model Schott KL1500; Zeiss, Jena, Germany) equipped with a Cy5-excitation filter (Chroma, Brattleboro, Vt., U.S.A.) and with two light guides whose extremities were placed at approximately 2 cm distance from the eye. Fluorescence was detected with a cooled C-5985 monochrome CCD-camera (Hamamatsu, Hamamatsu-City, Japan), equipped with C-mount Canon Zoom Lens (V6 ⁇ 16; 16-100 mm; 1:1.9) and a 50 mm diameter Cy5 emission filter (Chroma), placed at 3-4 cm distance from the irradiated eye. Acquisition times were 0.4 s.
  • Antibody fragments were in scFv format.
  • the purification of scFv(L19) and scFv(HyHEL-10) and their labeling with the N-hydroxysuccinimide (NHS) esters of indocyanine dyes have been described elsewhere [Neri, D. et al., Nature Biotechnol. 15, 1271-1275 (1997); Fattorusso, R., et al. (1999) Structure, 7, 381-390].
  • the Human Antibody Fragment L19 Chemically Conjugated to the Photosensitiser Sn (IV) Chlorine e6, Selectively Targets Ocular Angiogenesis and Mediates its Occlusion upon Irradiation with Red Light
  • FIG. 10 c Fluoroangiography with the indocyanine fluorophore Cy5 ( FIG. 10 c ) confirmed vessel occlusion as a characteristic hypofluorescent area. On the contrary, hyperfluorescent areas were observed in the leaky neovasculature of non-irradiated eyes ( FIG. 10 d,h ). No macroscopic alteration was detectable in the irradiated vessels of rabbits treated with ovalbumin-PS ( FIG. 10 e - g ), either ophthalmoscopically or by Cy5 fluoroangiography. The effect of irradiation of the targeted L19-PS conjugate at early stages of corneal angiogenesis are shown in FIG. 10 i - l .
  • FIG. 10 c,g Apoptosis in the portion of the cornea targeted by the photosensitiser conjugate was clearly visible in the fluorescent TUNEL assay ( FIG. 10 c,g ), but hardly detectable ink negative controls ( FIG. 10 d,h ).
  • FIG. 10 d,h A higher magnification view showed apoptosis of endothelial cells in vascular structures ( FIG. 10 g ). No damage to blood vessels of the iris, sclera and conjunctiva of treated animals could be observed either by TUNEL assay (not shown) or by H/E staining ( FIG. 10 k,l ).
  • Tin (IV) chlorin e 6 was selected from a panel of photosensitisers, on the basis of their potency, solubility and specificity, after coupling to a rabbit anti-mouse polyclonal antibody (Sigma). These immunoconjugates were screened by targeted photolysis of red blood cells coated with a monoclonal antibody specific for human CD47 (#313441A; Pharmingen, San Diego Calif., U.S.A.). Tin (IV) chlorin e 6 was prepared as described [Lu, X. M. et al., J. Immunol. Methods 156, 85-99 (1992)].
  • tin (IV) chlorin e 6 (2 mg/ml) was mixed for 30 min at room temperature in dimethylformamide with a ten-fold molar excess of EDC (N′-3-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride, Sigma) and NHS(N-hydoxysuccinimide, Sigma). The resulting activated mixture was then added to an eight-fold larger volume of protein solution (1 mg/ml) and incubated at room temperature for 1 h.
  • EDC N′-3-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride, Sigma
  • NHS(N-hydoxysuccinimide, Sigma) NHS(N-hydoxysuccinimide
  • antibody conjugates were separated from unincorporated fluorophore or photosensitiser using PD-10 columns (Amersham Pharmacia Biotech) equilibrated in 50 mM phosphate, pH 7.4, 100 mM NaCl (PBS). Immunoreactivity of antibody conjugates was measured as described in the previous Example.
  • rabbits were injected intravenously with 12 mg scFv(L19) 1 -tin (IV) chlorin e6 0.8 or 38 mg ovalbumin 1 -tin (IV) chlorin e6 0.36 , and kept in the dark for the duration of the experiment.
  • rabbits were anesthesised with ketamin (35 mg/kg)/xylazine (5 mg/kg)/acepromazin (1 mg/kg), and one of the two eyes was irradiated for 13 min with a Schott KL1500 tungsten halogen lamp equipped with a Cy5 filter (Chroma) and with two light guides whose extremities were placed at 1 cm distance from the eye.
  • the illuminated area was approximately 1 cm 2 , with an irradiation power density of 100 mW/cm 2 , measured using a SL818 photodetector (Newport Corp., Irvine, Calif., U.S.A.). No sign of animal discomfort after irradiation was observed.
  • rabbits received analgesics after irradiation (buprenorphine 0.03 mg/Kg).
  • KOWA SL-14 GMP SA, Rennens, Lausanne, Switzerland.
  • trimethylstannyl chloride (1.09 mg, 5.47 mmol) dissolved in 10 ml dry THF is added over 20 min to the reaction mixture.
  • the cooling bath is removed and the reaction is slowly brought to room temperature (RT) and stifling is kept for 1 hour.
  • the reaction is quenched by addition of water (10 ml) and extracted three times with 100 ml of diethyl ether.
  • the organic phase is washed with 5% NaHCO 3 (2 ⁇ 25 ml) and water (2 ⁇ 20 ml). After drying over MgSO 4 , it is concentrated on a rotary evaporator.
  • the product N-succinimidyl 3- 211 At-benzoate is isolated in 30% ethylacetate in Hexane (ca. 15 ml).
  • the radionuclide incorporation in the bifunctional agent m-MeATE is measured using an automated gamma counter with an energy window set to include the Polonium K X-rays emitted in the decay of 211 At.
  • Antibody immunoreactivity after labeling is evaluated by loading an aliquot of radiolabeled sample onto 200 ⁇ l of ED-B-Sepharose resin (capacity, >2.5 mg ED-B/ml resin) on a pasteur pipette, followed by radioactive counting of the flow-through and eluate fractions. Immunoreactivity, defined as the ratio between the counts of the eluted protein and the sum of the counts of the eluted and flow-through fractions, is >80%.
  • A2 SYA AISGSG GLSI Y G NGWYPW G4 SYA AISGSG SFSF Y G GGWLPY E1 SYA AISGSG FPFY Y G TGRIPP H10 SFS SIRGSS FPFY Y G TGRIPP L19 SFS SIRGSS FPFY Y Y TGRIPP
  • k off values from BIAcore experiments are not sufficietly reliable due to effects of the negatively-charged carboxylated solid dextran matrix; Kd values are therefore calculated from k off measurements obtained by competition experiments (Experimental Procedures).
  • K off kinetic dissociation constant
  • k on kinetic assocation constant
  • K d dissociation constant.
  • B measured on the BIAcore
  • C measured by competition with electrochemiluminescent detection. Values are accurate to +/ ⁇ 50%, on the basis of the precision of concentration determinations.

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